PROJECT SUMMARY Gram-negative bacteria are increasingly challenging to combat because existing antibiotics struggle to reach their intracellular targets and face elimination by efflux pumps. This issue is particularly pressing for bacteria that establish a replication-permissive vacuole derived from the host's plasma membrane. Shielded by multiple layers of membranes, intracellular pathogens become inaccessible to traditional antibiotics. A fundamental gap persists in the current understanding of how bacterial pathogens subvert host membrane transport processes and continued existence of this gap impedes our understanding of mechanisms that bacterial pathogens use to coordinate virulence strategies. Our long-term goal is to address this gap by systematically unveiling the host pathways critical for infection of human lung macrophages by the bacterial pathogen Legionella pneumophila, the causative agent of a severe pneumonia known as Legionnaires' disease. Legionella infects lung macrophages and resists degradation by establishing and residing within a membrane-bound compartment known as the Legionella-containing vacuole. Initially derived from the host cell's plasma membrane, this vacuolar membrane is dramatically remodeled during infection. To do so, the bacterium immediately begins translocating a large number of (effector) proteins directly into the host cytosol. The host membrane trafficking network is a major target of L. pneumophila effector proteins. In particular, vesicles traveling between the endoplasmic reticulum and the Golgi are sequestered by the Legionella-containing vacuole early during infection, whereas fusion with degradative lysosomes is prevented. These observations support the working model that the pathogen orchestrates its molecular interactions with the host to stimulate or inhibit fusion of host vesicles with its vacuole. Delineating the spatiotemporal distribution of secreted effectors is a critical step to understanding how L. pneumophila interacts with the host cell to ensure its own survival. The overall objective is to examine the spatiotemporal localization of L. pneumophila effector proteins in the context of human macrophage infection and to determine how L. pneumophila effectors interact with host phosphoinositide lipids to target membrane compartments. We propose: (1) to use a dual pronged approach based on chemical biology to directly track localization of L. pneumophila effectors in infected human macrophages, and (2) to characterize the protein-lipid interface between L. pneumophila effectors identified in our preliminary screen using X-ray crystallography. The proposed research is significant because it is positioned to advance our understanding of how bacterial pathogens manipulate host membrane transport pathways to promote intracellular survival of bacteria. A significant collateral outcome is that these studies could suggest new molecular targets for intervention in L. pneumophila infections and related conditions.